The present invention relates to a method for producing natural PUFA-enriched PUFA-triglyceride mixtures (PUFAs=Polyunsaturated Fatty Acids) having a minimum PUFA content of >55% by weight of TFA (Total Fatty Acids), the great majority of these consisting of triglycerides. These are obtained by winterization in one or more organic solvents from natural PUFA oils having a PUFA content>39% by weight of TFA.
Polyunsaturated fatty acids are essential fatty acids for the human organism. PUFAs can be subdivided into two large groups. In addition to the group of the ω-6 PUFAs, which are formulated starting from linoleic acid (18:2), there is the group of the ω-3 PUFAs, which are made up starting from α-linolenic acid (18:3.). PUFAs are important building blocks of cell membranes, the retina and the meninges, and are precursors for important hormones, for example prostaglandins, thromboxanes and leukotrienes.
Examples of nutritionally important PUFAs are shown in table 1:
TABLE 1Chain lengthand numberof C-CMost importantNamedouble bondssourcesOmega-3 fatty acidsα-Linolenic acidC 18:3linseed oil, soybean(ALA)oil, rapeseed oilStearidonic acidC 18:4microbially produced(SA)oilsEicosapentaenoicC 20:5saltwater fishacid(mackerel, salmon,(EPA)herring, sardine,tuna)DocosapentaenoicC 22:5saltwater fishacid(mackerel, salmon,(DPA)herring), microbiallyproduced oilsDocosahexaenoic acidC 22:6saltwater fish,(DHA)microbially producedoils (protists)Omega-6 fatty acidsγ-Linolenic acidC 18:3evening primrose oil,(GLA)borage oil,blackcurrantsArachidonic acidC 20:4saltwater fish,(ARA)microbially producedoils (Mortierella)
In addition to their function as building blocks, in recent years it has been found-that PUFAs directly have many beneficial effects on the human organism, or disorders.
A multiplicity of clinical studies have found that PUFAs, in the case of, for example, cancer, rheumatoid arthritis, high blood pressure and neurodermatitis and many other disorders, can make an important contribution to healing or relief. These results were originally responsible for international institutions and authorities making recommendations which control the daily intake of PUFAs.
PUFAs cannot be synthesized de novo by humans, since they lack the enzyme systems which could introduce a C—C double bond into the carbon chain at positions >C9 (lack of Δ12-desaturase). Not until precursor fatty acids (for example α-linolenic acid) are supplied via the diet are humans able to synthesize polyunsaturated fatty acids. However, whether this amount is sufficient to cover the requirement of polyunsaturated fatty acids is controversial.
The majority of the essential fatty acids is consumed via the diet. Plant oils in particular are enriched, with ω-6 fatty acids (for example blackcurrant contains GLA, that is to say gamma-linolenic acid), but these fatty acids are present here only in a chain length up to C18.
Fish oils contain ω-3 fatty acids (for example salmon oil contains EPA at up to 18% by weight of TFA, and DHA at 12% by weight of TFA). However, generally, the content of the desired PUFA is low and it is present in a mixture, in which case PUFAs having an antagonistic action can likewise be present. In particular in infant nutrition, EPA, owing to its bleeding-inhibition and growth-inhibition properties is undesirable (M. Hamosh (1998). Long-chain polyunsaturated fatty acids: who needs them? Biochem. Soc. Trans., 26(2), 96-103). There are natural limits to highly concentrated PUFA triglycerides from fish oils, on account of the triglyceride composition. The typical total content of EPA and DHA from fish oils is approximately 10-25% by weight of TFA. Owing to the multiplicity of different fatty acids and thus also varied triglyceride species, the maximum achievable PUFA concentrations are at most 30% by weight of TFA.
By using biocatalytic methods (employing lipases) it is possible to increase the. PUFA concentration in triglycerides to the range around DHA 40% by weight of TFA (Marinol D40® from Loders & Crocklaan, Wormerveer, Netherlands). However, natural oils cannot be produced in this manner.
A similar approach is the retrosynthesis of PUFA triglycerides or structural lipids. Here, by means of a lipase (specific or non-specific), high-purity PUFA triglycerides or structural lipids are produced starting from pure PUFAs (G. G. Haraldsson, B. Ö. Gudmundsson and Ö. Almarsson (1995): The synthesis of homogenous triglycerides of eicosapentaenoic acid and docosahexaenoic acid by lipase. Tetrahedron, 51(3), 941-952, R. Irimescu, K. Hata, Y. Iwasaki and T. Yamane (2001): Comparison of acyl donors for lipase-catalyzed production of 1,3-Dicapryloyl-2-eicosapentaenoylglycerol. JAOCS, 78(1), 65-67, F.-C. Huang, Y.-H. Ju and J.-C. Chiang (1999): γ-Linolenic acid-rich triacylglycerols derived from borrage oil via lipase-catalyzed reactions. JAOCS, 76(7), 833-837). The high cost of pure PUFAs, however, does not make this process appear very expedient. In these cases, also, natural oils are no longer present.
Producing relatively highly enriched PUFA concentrates (EPA+DHA>30% by weight. of TFA) in the form of natural triglycerides is therefore currently a challenge which has not been met (Haraldsson, G., G. (2000): Enrichement of Lipids with EPA and DHA by lipase. In Enzymes in Lipid Modification. Ed. U. Bornscheuer, Wiley VCH).
Producing relatively highly concentrated PUFA-triglyceride mixtures from marine oils is also difficult (S.-B. Park, Y. Endo, K. Maruyama and K. Fujimoto (2000): Enzymatic synthesis of ethyl ester of highly unsaturated fatty acids from fish oils using immobilized lipase. Food Sci. Technol., 6(3), 192-195). By means of complex chromatographic methods or short-path distillation, triglyceride fractions which are highly enriched with PUFAs may theoretically be isolated, but many problems occur (W. M. Willis, R. W. Lencki and A. G. Marangoni (1998): Lipid modification strategies in the production of nutritionally functional fats and oils. Crit. Rev. Food Sci., 38(8), 639-674, Hayashi, K. and H. Kishimira (1996): Preparation and purification of DHA-enriched triacylglycerols from fish oils by column chromatography. Fisheries Science, 62(5), 842-843). These methods are expensive and complex. Furthermore, a thermal load of the PUFAs which are labile to oxidation is undesirable and leads to product decomposition. Generally, these methods therefore lead to oils which are no longer natural.
Therefore, to produce PUFA concentrates, it is not natural oils which are used, but rather fatty acid or ester mixtures which are enriched, for example, by urea precipitation (S. P. J. N. Senanyake and F. Shahidi (2000): Concentration of docosahexaenoic acid (DHA) from algael oil via urea complexation. J. of Food Lipids, 7, 51-61, W. M. N. Ratnayake, B. Olsson, D. Matthews and R. G. Ackmann (1988): Preparation of omega-3 PUFA concentrates from fish oil via urea complexation. Fat. Sci. Technol., 10, 381-386). In these cases, very pure PUFA concentrates may likewise be produced. However, precipitation with urea is not suitable for triglycerides. Furthermore, the FDA has reported a physiologically hazardous formation of carbamate (carcinogenic class of substances) in the precipitation with urea (B. J. Canas (1999): Ethyl carbamate formation during urea complexation, for fractionation of fatty acids. JAOCS, 76(4), 537).
Other authors report small increases in PUFA concentration and poor yields according to processes such as dry fractionation (without solvent) or solvent crystallization (specifically with acetone). Bimbo and Crowther achieved an increase in DHA concentration from 10% by weight of TFA to 11% by weight of TFA and Lee et al. were able to achieve an increase in DHA/EPA concentration from 30.4% by weight of TFA to 35.3% by weight of TFA (A. P. Bimbo and J. B. Crowther (1991): Fish oils: processing beyond crude oil. Infofish International, 6, 20-25, K.-T. Lee and T. A. Foglia (2001): Fractionation of menhaden oil and partially hydrogenated menhaden oil: characterization of triacylglycerol fractions. JAOCS, 78(3), 297-303).
With a nominal amount of one DHA or EPA molecule per triglyceride, increasing the PUFA concentration without using enzymatic methods or resynthesis is not practicable above a concentration of 300 mg/g of oil. Achieving an EPA or DHA concentration of 300 mg/g of oil is not simple (Ackmann, R. A. (1988): The year of the fish oils. Chemistry and Industry, 7, 139-145). Higher contents are generally only offered in the form of the ethyl or methyl esters, although here also contents of a main PUFA of greater than 32.6% by weight of TFA are not achievable (Moffat, C. F., A. S. McGill, R. Hardy and R. A. Anderson (1993): The production of fish oils enriched in polyunsaturated fatty acid-containing triglycerides. JAOCS, 70(2), 133-138). Here, a complex method is used, in the form of freezing with liquid nitrogen and subsequent extraction at −60° C. The method is seen by the authors as a method for separating off solid triglycerides, rather than a method for producing highly enriched PUFA triglycerides. Liquid nitrogen is a dangerous substance which cannot be used simply, in particular industrially.
Only by using microorganisms is it possible to produce natural PUFA oils which have higher concentrations of PUFAs than fish oils or plant oils (K. D. Mukherjee (1999): Production and use of microbial oils. Inform, 10(4), 308-313). For instance, these oils can contain DHA at up to 40% by weight of TFA, GLA at 30% by weight of TFA, or ARA at 40% by weight of TFA.
For use in the food sector, in the clinical sector and in infant nutrition, the administration of natural triglycerides highly enriched with PUFAs is desirable, since it has been found that the intake of PUFAs in the form of triglycerides is particularly preferred (G. G. Haraldsson and A. Thorerensen (1999): Preparation of phospholipids highly enriched with n-3 polyunsaturated fatty acids by lipase. JAOCS, 76(10), 1143-1149, K. Osada, K. Takahashi and M. Hatano (1990): Hydrolysis and synthesis of icosapentaenoic acid-docosahexaenoic acid rich oil by lipase toyo. J. Jpn. Oil Chem. Soc., 1, 50-52). The use of free fatty acids in nutrition is unsuitable, in contrast,. In particular this relates to infant nutrition, since PUFAs likewise occur in human milk in the form of natural triglyceride mixtures (A. R. Medina, L. E. Cerdan, A. G. Gimenez, B. C. Paez, M. J. I. Gonzalez and E. M. Grima (1999): Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils. J. of Biotechnology, 70, 379-391).
Furthermore, the amount of PUFAs to be consumed daily is a problem, since high oil quantities (for example fish oils) need to be consumed. In particular, this relates to those patients who need to consume high concentrations of PUFAs (for example in the case of cystic fibrosis), since when high amounts of fish oil are consumed, side effects can occur (Y. Kosugi and A. Azuma (1994): Synthesis of triacylglycerols from polyunsaturated fatty acid by immobilized lipase, JAOCS, 71(12), 1397-1403). To achieve as specific an action as possible of the individual PUFAs, enriched or high-purity PUFAs must be used. There is therefore in the prior art a great requirement for natural triglycerides which are highly enriched with PUFAs (Haraldsson, G., G. (2000): Enrichement of Lipids with EPA and DHA by lipase. In Enzymes in Lipid Modification, Ed. U. Bornscheuer, Wiley VCH).
Winterization which has long been known is a method by which oils are separated off from solid fractions (waxes, fatty alcohols, saturated triglycerides) (from Wolf Hamm and Richard J. Hamilton (2000): Edible oil processing, Chapter 4, Sheffield Academic Press), or of producing oils having defined melting points (S. Hashimoto, T. Nezu, H. Arakawa, T. Ito and S. Maruzeni (2001): Preparation of sharp-melting hard palm midfraction and its use as hard butter in chocolate. JAOCS, 78(5), 455-460). In practice, this is the method of choice for producing oils from fats.
Yokochi et al. (1990) JAOCS, 67(11), 846-851, describe winterization for increasing the GLA concentration starting from an oil which was produced by fermentation with Mortierella ramanniana. However, Yokochi et al. do not achieve by far the PUFA concentrations which can be achieved using the present invention. GLA concentrations of only 10.5% by weight of TFA (GLA starting concentration 5.7% by weight of TFA) were achieved. In this method some solvents which are not permitted by food regulations were used (petroleum ether).
Linseed oil contains α-linolenic acid (18:3) at up to 62% by weight of TFA. No other natural composition is known in which another PUFA, in particular DHA and/or EPA, is present in such a high concentration in the form of triglycerides. In particular, however, other PUFAs such as DHA and/or EPA are also particularly beneficial to health. These PUFAs have more than 3 C—C double bonds.
Therefore, in the prior art, there is still a very great requirement for those compositions which contain natural PUFA triglycerides in high concentrations. There is a particularly great requirement for the following PUFAs: stearidonic acid (SA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), γ-linolenic acid (GLA) and arachidonic acid (ARA). There is a very particularly high requirement for eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and γ-linolenic acid (GLA).